Nanoparticles have unique properties that have been exploited for use in the delivery of DNA to cells. Among all nanoparticles investigated gold (Au) nanoparticles tend to be excellent candidates for delivery because of their low cytotoxicity and ease of functionalization with various ligands of biological significance. The commonly used synthesis of Au nanoparticles yields negatively charged (e.g., citrate coating) surface. Plasmid DNA, which may be sufficiently flexible to partially uncoil its bases, can be exposed to gold nanoparticles (“GNPs”). Under these partially uncoiled conditions, the negative charge on the DNA backbone may be sufficiently distant so that attractive van der Waals forces between the bases and the gold nanoparticle are sufficient to cause plasmid DNA to be attached to the surface of the gold particle.
In addition to metal nanoparticles, semi-conductor nanoparticles (e.g., quantum dots) (“QD”) within the size range of 3-5 nm have also been used as carriers to deliver molecules into cells. DNA and proteins can be linked to the ligand attached to the QD surface (see, e.g.,. Patolsky, F., et al., J. Am. Chem. Soc. 125, 13918 (2003)). Carboxylic acid or amine coated QDs can be cross linked to molecules containing a thiol group see, e.g., Dubertret B. et al., Science 298, 1759 (2002); Akerman, M. E., W. C. W. Chan, P. Laakkonen, S, N. Bhatia, E. Ruoslahti, Proc. Natl. Acad. Sci. U.S.A. 99, 12617 (2002); Mitchell, G. P., C. A. Mirkin, R. L. Letsinger, J. Am. Chem. Soc. 121, 8122 (1999) or an N-hydroxysuccinimyl (NHS)ester group by using standard bioconjugation protocols (see, e.g., Pinaud, F., D. King, H.-P. Moore, S. Weiss, J. Am. Chem. Soc. 126, 6115 (2004); Bruchez, M., M. Moronne, P. Gin, S. Weiss, A. P. Alivisatos, Science 281, 2013 (1998)). An alternative way is conjugation of streptavidin coated QDs to biotinylated proteins, oligos or antibodies (see, e.g., Dahan M. et al., Science 302, 442 (2003); Pinaud, F., D. King, H.-P. Moore, S. Weiss, J. Am. Chem. Soc. 126, 6115 (2004); Dahan M. et al., Science 302, 442 (2003); Wu. X. Y., et al., Nature Biotechnol. 21, 41 (2003); Jaiswal, J. K., H. Mattoussi, J. M. Mauro, S. M. Simon, Nature Biotechnol. 21, 47 (2003); and Mansson, A., et al., Biochem. Biophys. Res. Commun. 314, 529 (2004)).
Nanoparticles have been used to deliver plasmid DNA to a variety of animal cells. It has been found that when DNA coated nanoparticles are incubated with cells not having a cell wall, the cells take up the nanoparticles and begin expressing any genes encoded on the DNA. Where nanoparticle delivery to cells normally having a cell wall is desired, the cells wall is stripped before the addition of the particles to protoplasts of plant (see, Torrey, F. et al., Nature Nanotechnol. 2, (2007). In plant cells, the cell wall stands as a barrier for the delivery of exogenously applied molecules. Many invasive methods, like gene gun (biolistics), microinjection, electroporation, and Agrobacterium, have been employed to achieve gene and small molecule delivery into these walled plant cells, but delivery of proteins have only been achieved by microinjection. Delivery of small molecules and proteins in the presence of a cell wall of a plant cell remains unexplored and would be advantageous in order to develop enabling technologies to be deployed in intact plant cell/tissue or organ for in vitro and in vivo manipulations
The present invention relates to methods using nanoparticles to non-invasively deliver molecular substances into cells having a cell wall.